Bottom Line:
Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACTAlthough Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

fig1: Isolation of IgM-binding subclones and identification of cDNA inserts. (A) Cells transduced by the retroviral expression construct containing CLL-derived (top) or PMA-activated 697 pre–B cell–derived (bottom) cDNA libraries were enriched for IgM binding by FACS and subcloned for limiting dilution. Three representative subclones from each library are shown for their IgM-binding activity or lack of binding, as determined by flow cytometry. (B) Agarose gel electrophoresis analysis of RT-PCR products. RNA isolated from nontransduced control BW5147 T cells (lane 1) and from IgM-binding (lanes 3–5 and 7–9) or IgM-nonbinding (lanes 2 and 6) subclones from CLL-derived (lanes 2–5) and PMA-activated 697 pre–B cell–derived (lanes 6–9) cDNA libraries were subjected to RT-PCR as described in Materials and methods. Amplified products were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 10 is a PCR control without a first-strand cDNA template. HindIII-digested λ DNA was used as a size marker. The experiments were performed once for A and twice for B.

Mentions:
Our previous cellular and biochemical studies provided strong evidence for the existence of an FcμR that is expressed constitutively on chronic lymphocytic leukemia (CLL) B cells and inducibly on pre–B cell lines (Sanders et al., 1987; Ohno et al., 1990). To identify the gene encoding the putative FcμR, two different cDNA libraries from CLL B cells and a PMA-activated 697 pre–B cell line were constructed in a retroviral expression vector and then introduced into mouse T cell line BW5147. Transduced cells exhibiting IgM binding were enriched by FACS and subcloned. Many of the single cell–derived subclones from both cDNA libraries bound IgM (Fig. 1 A). RT-PCR analysis revealed that a DNA fragment of ∼2 kb was specifically amplified only from IgM-binding subclones (Fig. 1 B), and their nucleotide sequence analyses defined an identical 1,173-bp open reading frame (CLL- and PMA-activated 697 pre–B cell–derived FcμR cDNA available from GenBank/EMBL/DDBJ under accession nos. GQ160900 and GQ160901, respectively; Fig. S1). Basic local alignment search technique database analysis revealed that the isolated FcμR cDNA was identical to that of the previously described human Fas apoptotic inhibitory molecule 3 (FAIM3; available from GenBank/EMBL/DDBJ under accession no. NM_005449), except for one nucleotide difference at a position reported as a synonymous single nucleotide polymorphism. FAIM3 was identified in a similar retroviral cDNA library–based functional assay as a potent inhibitor of Fas/CD95-induced apoptotic signaling and was originally designated as TOSO, after a Japanese liquor drunk on New Year’s day to celebrate long life and eternal youth (Hitoshi et al., 1998). Interestingly, however, the apoptosis in this functional assay was induced by ligation of Fas with a mouse mAb of IgM isotype (CH11). This immediately raised the possibility that the CH11 mAb bound to the Fas via its Fabμ portion and to the FAIM3/TOSO via its Fcμ portion, thereby bringing them in close physical proximity in a process reminiscent of that described in FcγRIIb-mediated inhibition of BCR signaling by intact IgG anti-μ antibodies (Tony and Schimpl, 1980; Ravetch and Nimmerjahn, 2008).

fig1: Isolation of IgM-binding subclones and identification of cDNA inserts. (A) Cells transduced by the retroviral expression construct containing CLL-derived (top) or PMA-activated 697 pre–B cell–derived (bottom) cDNA libraries were enriched for IgM binding by FACS and subcloned for limiting dilution. Three representative subclones from each library are shown for their IgM-binding activity or lack of binding, as determined by flow cytometry. (B) Agarose gel electrophoresis analysis of RT-PCR products. RNA isolated from nontransduced control BW5147 T cells (lane 1) and from IgM-binding (lanes 3–5 and 7–9) or IgM-nonbinding (lanes 2 and 6) subclones from CLL-derived (lanes 2–5) and PMA-activated 697 pre–B cell–derived (lanes 6–9) cDNA libraries were subjected to RT-PCR as described in Materials and methods. Amplified products were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 10 is a PCR control without a first-strand cDNA template. HindIII-digested λ DNA was used as a size marker. The experiments were performed once for A and twice for B.

Mentions:
Our previous cellular and biochemical studies provided strong evidence for the existence of an FcμR that is expressed constitutively on chronic lymphocytic leukemia (CLL) B cells and inducibly on pre–B cell lines (Sanders et al., 1987; Ohno et al., 1990). To identify the gene encoding the putative FcμR, two different cDNA libraries from CLL B cells and a PMA-activated 697 pre–B cell line were constructed in a retroviral expression vector and then introduced into mouse T cell line BW5147. Transduced cells exhibiting IgM binding were enriched by FACS and subcloned. Many of the single cell–derived subclones from both cDNA libraries bound IgM (Fig. 1 A). RT-PCR analysis revealed that a DNA fragment of ∼2 kb was specifically amplified only from IgM-binding subclones (Fig. 1 B), and their nucleotide sequence analyses defined an identical 1,173-bp open reading frame (CLL- and PMA-activated 697 pre–B cell–derived FcμR cDNA available from GenBank/EMBL/DDBJ under accession nos. GQ160900 and GQ160901, respectively; Fig. S1). Basic local alignment search technique database analysis revealed that the isolated FcμR cDNA was identical to that of the previously described human Fas apoptotic inhibitory molecule 3 (FAIM3; available from GenBank/EMBL/DDBJ under accession no. NM_005449), except for one nucleotide difference at a position reported as a synonymous single nucleotide polymorphism. FAIM3 was identified in a similar retroviral cDNA library–based functional assay as a potent inhibitor of Fas/CD95-induced apoptotic signaling and was originally designated as TOSO, after a Japanese liquor drunk on New Year’s day to celebrate long life and eternal youth (Hitoshi et al., 1998). Interestingly, however, the apoptosis in this functional assay was induced by ligation of Fas with a mouse mAb of IgM isotype (CH11). This immediately raised the possibility that the CH11 mAb bound to the Fas via its Fabμ portion and to the FAIM3/TOSO via its Fcμ portion, thereby bringing them in close physical proximity in a process reminiscent of that described in FcγRIIb-mediated inhibition of BCR signaling by intact IgG anti-μ antibodies (Tony and Schimpl, 1980; Ravetch and Nimmerjahn, 2008).

Bottom Line:
Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes.After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation.Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Affiliation:
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACTAlthough Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.